Antibody complexes and methods for immunolabeling

• US 8,535,894 B2
• Filed: 09/11/2012
• Issued: 09/17/2013
• Est. Priority Date: 10/12/2001
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Archer, Robert, Haugland, Richard, Haugland, Rosaria, Beechem, Joseph, Hagen, David
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US13/610,009
• Publication Number:
  US 20130072666A1
• Time in Patent Office: 371 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor

Abstract

Provided are labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents. The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label. Discrete subsets of labeling reagent and immune-labeled complexes are provided that facilitate the simultaneous detection of multiple targets in a sample-complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody. This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets.

Claims

1. A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of:

a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments;

b) isolating said anti-region Fab fragments of step a);

c) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said anti-region Fab fragments are immobilized on said matrix;

d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group;

e) washing said matrix of step d) to remove unbound label, and;

f) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured.

2. The method according to claim 1, wherein said anti-region Fab fragment are selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.

3. The method according to claim 2, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.

4. A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of:

a) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized on said matrix;

b) contacting said matrix of step a) with a solution comprising a fluorophore label that contains a reactive group;

c) washing said matrix to remove unbound label, and;

d) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured that comprises a fluorophore label.

5. The method according to claim 4, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.

6. The method according to claim 5, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.

7. An isolated labeling reagent made by a process comprising:

a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments;

b) isolating said anti-region Fab fragments of step a);

c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region Fab fragments of step b) wherein said anti-region Fab fragments are immobilized;

d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group;

e) washing said matrix to remove unbound label, and;

f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins or fragments thereof.

8. The labeling reagent according to claim 7, wherein said anti-region Fab fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.

9. The labeling reagent according to claim 8, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.